Picornaviruses are among the largest of animal virus families and include cardio-, polio-, rhino-, and hepatitis A viruses. The objective of this proposal is to examine three early stages of picornavirus infection: the interaction with the cellular receptor, the receptor-mediated conformational changes in the virion, and the viral usurping of normal cellular functions via a number of non-capsid proteins. The dominant dogma is that some of these viruses have evolved 'canyon' regions to protect the receptor-binding region from immune surveillance and that host-derived 'pocket factors' bind under the canyons to modulate receptor interactions. Our recent results are inconsistent with these premises and led us to propose an alternative hypothesis: We propose that the canyon is not the evolutionary consequence of immunological pressure but exists to facilitate capsid dynamics and 'pocket factors' are probably an artifact. This grant will directly test these opposing hypotheses by addressing the following questions: 1) Has immunity shaped the capsid structure and how is viral entry blocked by antibodies? We will continue structural studies on antibody/virus complexes since they offer details necessary for future vaccine development and the true function of the canyon. We will also make heavy-chain-only antibody libraries to directly test aspects of the canyon hypothesis and offer a novel way to find the 'Achilles heel' of a virus. 2) What are the structural details of the capsid dynamics and do 'pocket factors' play a role? In our recent work, we found that the HRV14 viral capsid is remarkably dynamic. We will use molecular biology, mass spectroscopy, and cryo-TEM to further examine the details of this capsid 'breathing'. These studies not only will elucidate what we believe to be the true function of the canyon, but will also yield insight as to how the virus gets its genome into the host cell. 3) What are the structures of the non-capsid proteins and how do they usurp cellular function? Picornaviruses differ in aspects of the early stages of replication and how they usurp normal cell functions. We will determine the structures of some of these proteins to better understand this process and how the replication complexes are assembled. These proteins are also natural targets for antiviral agents. These studies have the potential of changing the way we view the early steps in the viral infection process and will allow us to develop tools that are applicable to other viral systems.